High Resolution Label Free Analysis of Cellular Properties

ABSTRACT

The invention provides methods of detecting a change in cell growth patterns.

PRIORITY

This application claims the benefit of U.S. Ser. No. 61/043,478, filed on Apr. 9, 2008, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

It has been estimated that at least two days of laboratory time and the use of fluorescent labels are required to assess cellular changes upon exposure to biological entities. See, e.g., Dharmawardhane et al., 1997, J. Cell Biol. 138(6):1265-78. Additionally, it has been estimated that at least 8-24 hours of laboratory time and the use of a secondary dye are required to quantify total cell movement or cell changes toward biological entities, such as a protein, peptide or small molecule. See, Reckless & Grainger. 1999. Biochem. J. 340: 803-811, Taguchi et al. 1998. J. Exp. Med. 187(12): 1927-1940, Jackson et al. 1999. J. Pharm. & Exper. Therapeutics. 288(1): 286-294 and Yarrow et al., 2004 BMC Biotechnol. 4(21):1-9. Methods are needed to reduce the time to perform these assays.

SUMMARY OF THE INVENTION

One embodiment of the invention provides a method of detecting a change in a cell growth pattern. The method comprises immobilizing one or more types of cells to a location on a surface of a calorimetric resonant reflectance optical biosensor with an antibody or a binding fragment thereof that specifically binds an adhesion protein; detecting a first calorimetric resonant reflectance optical peak wavelength value (PWV) for the location; incubating the one or more cells for a period of time or applying a test reagent to the one or more cells and incubating the one or more cells for a period of time; detecting a second calorimetric resonant reflectance optical PWV for the location; and comparing the first PWV to the second PWV. A difference between the first calorimetric resonant reflectance optical PWV in relation to the second calorimetric resonant reflectance optical PWV indicates a change in the cell growth pattern in the one or more cells. The change in cell growth pattern can be a change in cell morphology, change in cell adhesion, change in cell migration, change in cell proliferation, change in cell death, change in microtubule structure, change in microfilament structure, granule exocytosis, respiratory burst, cell differentiation, or a combination thereof. The PWVs can be detected using a scanner with a lens having a lower limit pixel size of about 1 micrometer to about 15 micrometers. The location on a surface of a calorimetric resonant reflectance optical biosensor can be an internal surface of a vessel selected from the group consisting of a microtiter well, microtiter plate, test tube, Petri dish, microfluidic channel, and microarray. The method can be completed in less than one hour.

Another embodiment of the invention provides a method of detecting a change in a cell growth pattern. The method comprises adding one or more types of cells to a calorimetric resonant reflectance optical biosensor, wherein one or more antibodies or a binding fragments thereof that specifically bind one or more adhesion proteins are immobilized on a location on the biosensor surface; optionally adding one or more adhesion proteins that specifically bind the one or more antibodies or binding fragments thereof to the biosensor surface; detecting a first calorimetric resonant reflectance optical peak wavelength value (PWV) for the location; incubating the one or more cells for a period of time or applying a test reagent to the one or more cells and incubating the one or more cells for a period of time; detecting a second PWV for the location; and comparing the first PWV and second PWV. A difference between the first PWV in relation to the second PWV indicates a change in the cell growth pattern in the one or more cells. The one or more cells can express an adhesion protein that specifically binds the one or more antibodies or binding fragments thereof. The one or more cells can express a receptor that specifically binds the one or more adhesion proteins.

Still another embodiment of the invention provides a method of detecting a change in a cell growth pattern. The method comprises adding one or more types of cells to a calorimetric resonant reflectance optical biosensor, wherein one or more antibodies or a binding fragments thereof that specifically bind one or more adhesion proteins are immobilized on a location on the biosensor surface; detecting calorimetric resonant reflectance optical peak wavelength values (PWVs) for the location over the whole time of the assay; optionally adding one or more adhesion proteins that specifically bind the one or more antibodies or binding fragments thereof to the biosensor surface; and incubating the one or more cells for a period of time or applying a test reagent to the one or more cells and incubating the one or more cells for a period of time. A difference between the PWVs over the time of the assay indicates a change in the cell growth pattern in the one or more cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a cross-sectional view of a colorimetric resonant reflectance biosensor wherein light is shown as illuminating the bottom of the biosensor; however, light can illuminate the biosensor from either the top or the bottom. FIG. 1B shows a diagram of a calorimetric resonant reflectance biosensor wherein light is shown as illuminating the bottom of the biosensor; however, light can illuminate the biosensor from either the top or the bottom;

FIG. 2 shows an embodiment of a calorimetric resonant reflection biosensor comprising a one-dimensional grating.

FIG. 3 shows a schematic diagram of a VLA-4 and VCAM-1 adhesion assay.

FIG. 4 shows the results from a VLA-4 and VCAM-1 adhesion assay.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a,” “an”, and “the” include plural referents unless the context clearly dictates otherwise.

One embodiment of the invention allows the direct detection of cell changes as they occur in real time with a calorimetric resonant reflectance biosensor and without the need to incorporate or without interference from radiometric, calorimetric, or fluorescent labels. Changes in cell behavior and morphology can be detected as the cell is perturbed. The cellular changes can then be detected in real time using a high speed, high resolution instrument, such as the BIND Scanner™ (i.e., a calorimetric resonant reflectance biosensor system), and corresponding algorithms to quantify data. See, e.g., U.S. Pat. No. 6,951,715 and U.S. Pat. Publ. 2004/0151626. By combining this methodology, instrumentation and computational analysis, cellular behavior can be expediently monitored in real time, in a label free manner.

Colorimetric resonant reflectance biosensors, such as SRU Biosystems, Inc. BIND™ technology (Woburn, Mass.) have the capability of measuring changes to a surface with respect to mass attachment from nanoscale biological systems. The applications and the methods, in which calorimetric resonant reflectance biosensors have been previously implemented, have changed as the resolution of the instruments has improved. Previously, measurement of the quantity of cells attached to the calorimetric resonant reflectance biosensor surface was the primary goal. While looking at some poorer resolution images of cells, however, it was noted that cells gave differential signals with respect to the number of pixels occupied, intensity of signal/pixel, change in PWV of each pixel, etc. While trying to reduce the variability of these data, it became clear that the variability lay within the individual cells and their differential morphological responses to stimuli. To further investigate these cellular events, a higher resolution version of a BIND Scanner™ (i.e., a calorimetric resonant reflectance biosensor system), was constructed. The scanner has a higher resolution lens than previously used scanners. The lens has a lower limit pixel size of about 7 micrometers. Additionally, a methodology was developed for analyzing cell changes in real time at better resolution.

Biosensors

Biosensors of the invention can be calorimetric resonant reflectance biosensors. See e.g., Cunningham et al., “Colorimetric resonant reflection as a direct biochemical assay technique,” Sensors and Actuators B, Volume 81, p. 316-328, Jan. 5, 2002; U.S. Pat. Publ. No. 2004/0091397. Colorimetric resonant biosensors are not surface plasmon resonant (SPR) biosensors. SPR biosensors have a thin metal layer, such as silver, gold, copper, aluminum, sodium, and indium. The metal must have conduction band electrons capable of resonating with light at a suitable wavelength. A SPR biosensor surface exposed to light must be pure metal. Oxides, sulfides and other films interfere with SPR. Colorimetric resonant biosensors do not have a metal layer, rather they have a dielectric coating of high refractive index material, such as TiO₂.

Grating-based waveguide biosensors are described in, e.g., U.S. Pat. No. 5,738,825. A grating-based waveguide biosensor comprises a waveguiding film and a diffraction grating that incouples an incident light field into the waveguiding film to generate a diffracted light field. A change in the effective refractive index of the waveguiding film is detected. Devices where the wave must be transported a significant distance within the device, such as grating-based waveguide biosensors, lack the spatial resolution of the current invention.

A calorimetric resonant reflectance biosensor allows biochemical interactions to be measured on the biosensor's surface without the use of fluorescent tags, calorimetric labels or any other type of detection tag or detection label. A biosensor surface contains an optical structure that, when illuminated with collimated and/or white light, is designed to reflect only a narrow band of wavelengths (“a resonant grating effect”). The narrow wavelength band (e.g., about 1 to about 10 nm) is described as a wavelength “peak.” The “peak wavelength value” (PWV) changes when materials, such as biological materials, are deposited or removed from the biosensor surface. A readout instrument is used to illuminate distinct locations on a biosensor surface with collimated and/or white light, and to collect reflected light. The collected light is gathered into a wavelength spectrometer for determination of a PWV.

A biosensor can be incorporated into standard disposable laboratory items such as microtiter plates by bonding the structure (biosensor side up) into the bottom of a bottomless microtiter plate cartridge. Incorporation of a biosensor into common laboratory format cartridges is desirable for compatibility with existing microtiter plate handling equipment such as mixers, incubators, and liquid dispensing equipment. Colorimetric resonant reflectance biosensors can also be incorporated into, e.g., microfluidic, macrofluidic, or microarray devices (see, e.g., U.S. Pat. No. 7,033,819, U.S. Pat. No. 7,033,821). Colorimetric resonant reflectance biosensors can be used with well-know methodology in the art (see, e.g., Methods of Molecular Biology edited by Jun-Lin Guan, Vol. 294, Humana Press, Totowa, N.J.) to monitor cell behavioral changes or the lack of these changes upon exposure to one or more extracellular reagents.

Colorimetric resonant reflectance biosensors comprise subwavelength structured surfaces (SWS) and are an unconventional type of diffractive optic that can mimic the effect of thin-film coatings. (Peng & Morris, “Resonant scattering from two-dimensional gratings,” J. Opt. Soc. Am. A, Vol. 13, No. 5, p. 993, May 1996; Magnusson, & Wang, “New principle for optical filters,” Appl. Phys. Lett., 61, No. 9, p. 1022, August, 1992; Peng & Morris, “Experimental demonstration of resonant anomalies in diffraction from two-dimensional gratings,” Optics Letters, Vol. 21, No. 8, p. 549, April, 1996). A SWS structure contains a one-dimensional, two-dimensional, or three dimensional grating in which the grating period is small compared to the wavelength of incident light so that no diffractive orders other than the reflected and transmitted zeroth orders are allowed to propagate. Propagation of guided modes in the lateral direction are not supported. Rather, the guided mode resonant effect occurs over a highly localized region of approximately 3 microns from the point that any photon enters the biosensor structure.

The reflected or transmitted light of a calorimetric resonant reflectance biosensor can be modulated by the addition of molecules such as specific binding substances or binding partners or both to the upper surface of the biosensor. The added molecules increase the optical path length of incident radiation through the structure, and thus modify the wavelength at which maximum reflectance or transmittance will occur.

In one embodiment, a calorimetric resonant reflectance biosensor, when illuminated with white and/or collimated light, is designed to reflect a single wavelength or a narrow band of wavelengths (a “resonant grating effect”). When mass is deposited on the surface of the biosensor, the reflected wavelength is shifted due to the change of the optical path of light that is shown on the biosensor.

A detection system consists of, for example, a light source that illuminates a small spot of a biosensor at normal incidence through, for example, a fiber optic probe, and a spectrometer that collects the reflected light through, for example, a second fiber optic probe also at normal incidence. Because no physical contact occurs between the excitation/detection system and the biosensor surface, no special coupling prisms are required and the biosensor can be easily adapted to any commonly used assay platform including, for example, microtiter plates. A single spectrometer reading can be performed in several milliseconds, thus it is possible to quickly measure a large number of molecular interactions taking place in parallel upon a biosensor surface, and to monitor reaction kinetics in real time.

FIGS. 1A and 1B are diagrams of an example of a calorimetric resonant reflectance biosensor. In FIG. 1, n_(substrate) represents a substrate material. n₂ represents the refractive index of an optical grating. n₁ represents an optional cover layer. n_(bio) represents the refractive index of an optional biological material. t₁ represents the thickness of the optional cover layer above the one-, two- or three-dimensional grating structure. t₂ represents the thickness of the grating. t_(bio) represents the thickness of the layer of the biological material. In one embodiment, are n2<n1 (see FIG. 1A). Layer thicknesses (i.e. cover layer, biological material, or an optical grating) are selected to achieve resonant wavelength sensitivity to additional molecules on the top surface. The grating period is selected to achieve resonance at a desired wavelength.

A calorimetric resonant reflectance biosensor comprises, e.g., an optical grating comprised of a high refractive index material, a substrate layer that supports the grating, and optionally one or more specific binding substances or linkers immobilized on the surface of the grating opposite of the substrate layer. The high refractive index material has a higher refractive index than a substrate layer. See, e.g., U.S. Pat. No. 7,094,595; U.S. Pat. No. 7,070,987. Optionally, a cover layer covers the grating surface. An optical grating is coated with a high refractive index dielectric film which can be comprised of a material that includes, for example, zinc sulfide, titanium dioxide, titanium oxide, titanium phosphate, tantalum oxide, silicon nitride, and silicon dioxide. A cross-sectional profile of a grating with optical features can comprise any periodically repeating function, for example, a “square-wave.” An optical grating can also comprise a repeating pattern of shapes selected from the group consisting of lines (one-dimensional), squares, circles, ellipses, triangles, trapezoids, sinusoidal waves, ovals, rectangles, and hexagons. A calorimetric resonant reflectance biosensor of the invention can also comprise an optical grating comprised of, for example, plastic or epoxy, which is coated with a high refractive index material.

Linear gratings (i.e., one dimensional gratings) have resonant characteristics where the illuminating light polarization is oriented perpendicular to the grating period. A schematic diagram of one embodiment a linear grating structure with an optional cover layer is shown in FIG. 2. A calorimetric resonant reflection biosensor can also comprise, for example, a two-dimensional grating, e.g., a hexagonal array of holes or squares. Other shapes can be used as well. A linear grating has the same pitch (i.e. distance between regions of high and low refractive index), period, layer thicknesses, and material properties as a hexagonal array grating. However, light must be polarized perpendicular to the grating lines in order to be resonantly coupled into the optical structure. Therefore, a polarizing filter oriented with its polarization axis perpendicular to the linear grating must be inserted between the illumination source and the biosensor surface. Because only a small portion of the illuminating light source is correctly polarized, a longer integration time is required to collect an equivalent amount of resonantly reflected light compared to a hexagonal grating.

An optical grating can also comprise, for example, a “stepped” profile, in which high refractive index regions of a single, fixed height are embedded within a lower refractive index cover layer. The alternating regions of high and low refractive index provide an optical waveguide parallel to the top surface of the biosensor.

A calorimetric resonant reflectance biosensor of the invention can further comprise a cover layer on the surface of an optical grating opposite of a substrate layer. Where a cover layer is present, the one or more specific binding substances are immobilized on the surface of the cover layer opposite of the grating. Preferably, a cover layer comprises a material that has a lower refractive index than a material that comprises the grating. A cover layer can be comprised of, for example, glass (including spin-on glass (SOG)), epoxy, or plastic.

For example, various polymers that meet the refractive index requirement of a biosensor can be used for a cover layer. SOG can be used due to its favorable refractive index, ease of handling, and readiness of being activated with specific binding substances using the wealth of glass surface activation techniques. When the flatness of the biosensor surface is not an issue for a particular system setup, a grating structure of SiN/glass can directly be used as the sensing surface, the activation of which can be done using the same means as on a glass surface.

Resonant reflection can also be obtained without a planarizing cover layer over an optical grating. For example, a biosensor can contain only a substrate coated with a structured thin film layer of high refractive index material. Without the use of a planarizing cover layer, the surrounding medium (such as air or water) fills the grating. Therefore, specific binding substances are immobilized to the biosensor on all surfaces of an optical grating exposed to the specific binding substances, rather than only on an upper surface.

In general, a colorimetric resonant reflectance biosensor of the invention will be illuminated with white and/or collimated light that will contain light of every polarization angle. The orientation of the polarization angle with respect to repeating features in a biosensor grating will determine the resonance wavelength. For example, a “linear grating” (i.e., a one-dimensional grating) biosensor consisting of a set of repeating lines and spaces will have two optical polarizations that can generate separate resonant reflections. Light that is polarized perpendicularly to the lines is called “s-polarized,” while light that is polarized parallel to the lines is called “p-polarized.” Both the s and p components of incident light exist simultaneously in an unfiltered illumination beam, and each generates a separate resonant signal. A biosensor can generally be designed to optimize the properties of only one polarization (the s-polarization), and the non-optimized polarization is easily removed by a polarizing filter.

In order to remove the polarization dependence, so that every polarization angle generates the same resonant reflection spectra, an alternate biosensor structure can be used that consists of a set of concentric rings. In this structure, the difference between the inside diameter and the outside diameter of each concentric ring is equal to about one-half of a grating period. Each successive ring has an inside diameter that is about one grating period greater than the inside diameter of the previous ring. The concentric ring pattern extends to cover a single sensor location—such as an array spot or a microtiter plate well. Each separate microarray spot or microtiter plate well has a separate concentric ring pattern centered within it. All polarization directions of such a structure have the same cross-sectional profile. The concentric ring structure must be illuminated precisely on-center to preserve polarization independence. The grating period of a concentric ring structure is less than the wavelength of the resonantly reflected light. The grating period is about 0.01 micron to about 1 micron. The grating depth is about 0.01 to about 1 micron.

In another embodiment, an array of holes or posts are arranged to closely approximate the concentric circle structure described above without requiring the illumination beam to be centered upon any particular location of the grid. Such an array pattern is automatically generated by the optical interference of three laser beams incident on a surface from three directions at equal angles. In this pattern, the holes (or posts) are centered upon the corners of an array of closely packed hexagons. The holes or posts also occur in the center of each hexagon. Such a hexagonal grid of holes or posts has three polarization directions that “see” the same cross-sectional profile. The hexagonal grid structure, therefore, provides equivalent resonant reflection spectra using light of any polarization angle. Thus, no polarizing filter is required to remove unwanted reflected signal components. The period of the holes or posts can be about 0.01 microns to about 1 micron and the depth or height can be about 0.01 microns to about 1 micron.

A detection system can comprise a calorimetric resonant reflectance biosensor a light source that directs light to the calorimetric resonant reflectance biosensor, and a detector that detects light reflected from the biosensor. In one embodiment, it is possible to simplify the readout instrumentation by the application of a filter so that only positive results over a determined threshold trigger a detection.

By measuring the shift in resonant wavelength at each distinct location of a calorimetric resonant reflectance biosensor of the invention, it is possible to determine which distinct locations have, e.g., biological material deposited on them. The extent of the shift can be used to determine, e.g., the amount of binding partners in a test sample and the chemical affinity between one or more specific binding substances and the binding partners of the test sample.

A calorimetric resonant reflectance biosensor can be illuminated twice. The first measurement determines the reflectance spectra of one or more distinct locations of a biosensor with, e.g., no biological material on the biosensor. The second measurement determines the reflectance spectra after, e.g., one or more cells are applied to a biosensor. The difference in peak wavelength between these two measurements is a measurement of the presence or amount of cells on the biosensor. This method of illumination can control for small imperfections in a surface of a biosensor that can result in regions with slight variations in the peak resonant wavelength. This method can also control for varying concentrations or density of cell matter on a biosensor.

Surface of Biosensor

One or more cells can be immobilized on a biosensor by for example, physical adsorption or by chemical binding. A cell can specifically bind to a biosensor surface via a specific binding substance such as a nucleic acid, peptide, an antibody or binding fragment thereof that specifically binds an adhesion protein, an adhesion protein, protein solution, peptide solution, solutions containing compounds from a combinatorial chemical library, antigen, polyclonal antibody, monoclonal antibody, single chain antibody (scFv), F(ab) fragment, F(ab′)₂ fragment, Fv fragment, small organic molecule, virus, polymer or biological sample, wherein the specific binding substance is immobilized to the surface of the biosensor and the binding partner is on the surface of the cell.

Furthermore, cells can be arranged in an array of one or more distinct locations on the biosensor surface, said surface residing within one or more wells of a multiwell plate and comprising one or more surfaces of the multiwell plate or microarray. The array of cells comprises one or more cells on the biosensor surface within a microwell plate such that a surface contains one or more distinct locations, each with a different cell or with a different amount of cells. For example, an array can comprise 1, 10, 100, 1,000, 10,000 or 100,000 or greater distinct locations. Thus, each well of a multiwell plate or microarray can have within it an array of one or more distinct locations separate from the other wells of the multiwell plate, which allows multiple different samples to be processed on one multiwell plate. The array or arrays within any one well can be the same or different than the array or arrays found in any other microtiter wells of the same microtiter plate.

Immobilization of a cell to a biosensor surface can be also be affected via binding to, for example, the following functional linkers: a nickel group, an amine group, an aldehyde group, an acid group, an alkane group, an alkene group, an alkyne group, an aromatic group, an alcohol group, an ether group, a ketone group, an ester group, an amide group, an amino acid group, a nitro group, a nitrile group, a carbohydrate group, a thiol group, an organic phosphate group, a lipid group, a phospholipid group or a steroid group. Furthermore, a cell can be immobilized on the surface of a biosensor via physical adsorption, chemical binding, electrochemical binding, electrostatic binding, hydrophobic binding or hydrophilic binding, and immunocapture methods.

In one embodiment of the invention a biosensor can be coated with a linker such as, e.g., a nickel group, an amine group, an aldehyde group, an acid group, an alkane group, an alkene group, an alkyne group, an aromatic group, an alcohol group, an ether group, a ketone group, an ester group, an amide group, an amino acid group, a nitro group, a nitrile group, a carbohydrate group, a thiol group, an organic phosphate group, a lipid group, a phospholipid group or a steroid group. For example, an amine surface can be used to attach several types of linker molecules while an aldehyde surface can be used to bind proteins directly, without an additional linker. A nickel surface can be used to bind molecules that have an incorporated histidine (“his”) tag. Detection of “his-tagged” molecules with a nickel-activated surface is well known in the art (Whitesides, Anal. Chem. 68, 490, (1996)).

Linkers and specific binding substances can be immobilized on the surface of a biosensor such that each well has the same linkers and/or specific binding substances immobilized therein. Alternatively, each well can contain a different combination of linkers and/or specific binding substances.

A cell can specifically or non-specifically bind to a linker or specific binding substance immobilized on the surface of a biosensor. Alternatively, the surface of the biosensor can have no linker or specific binding substance and a cell can bind to the biosensor surface non-specifically.

Immobilization of one or more specific binding substances or linker onto a biosensor is performed so that a specific binding substance or linker will not be washed away by rinsing procedures, and so that its binding to cells in a test sample is unimpeded by the biosensor surface. Several different types of surface chemistry strategies have been implemented for covalent attachment of specific binding substances to, for example, glass for use in various types of microarrays and biosensors. These same methods can be readily adapted to a biosensor of the invention. Surface preparation of a biosensor so that it contains the correct functional groups for binding one or more specific binding substances is an integral part of the biosensor manufacturing process.

One or more specific cells can be attached to a biosensor surface by physical adsorption (i.e., without the use of chemical linkers) or by chemical binding (i.e., with the use of chemical linkers) as well as electrochemical binding, electrostatic binding, hydrophobic binding and hydrophilic binding. Chemical binding can generate stronger attachment of specific binding substances on a biosensor surface and provide defined orientation and conformation of the surface-bound molecules.

Immobilization of specific binding substances to plastic, epoxy, or high refractive index material can be performed essentially as described for immobilization to glass. However, the acid wash step can be eliminated where such a treatment would damage the material to which the specific binding substances are immobilized.

Detecting Changes in Cell Growth Patterns or Cell Properties

It has been estimated that at least 8-24 hours of laboratory time and the use of a secondary dye are required to quantify total cell movement or cell changes in response to biological entities, such as a protein, peptide or small molecule. See, Reckless & Grainger. 1999. Biochem. J. 340: 803-811, Taguchi et al. 1998. J. Exp. Med. 187(12): 1927-1940, Jackson et al. 1999. J. Pharm. & Exper. Therapeutics. 288(1): 286-294 and Yarrow et al., 2004 BMC Biotechnol. 4(21):1-9; see also, U.S. Patent Appl. 2003/0068657, U.S. Patent Appl. 2003/0108954, U.S. Patent Appl. 2004/0091397, U.S. Patent Appl. 2005/0221271, U.S. Patent Appl. 2005/0074825, U.S. Patent Appl. 2005/0058639, U.S. Pat. No. 7,018,838, U.S. Pat. No. 6,982,171, and U.S. Pat. No. 5,601,997. The required amount of time for these types of assays can be reduced to a maximum of 3 hours or less using methods and compositions of the invention. For example, depending on the length of time cells are allowed to incubate on the surface of the biosensor, an assay can be completed in less than about 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 10 minutes, 5 minutes or 3 minutes. Additionally, no dyes or detection labels are necessary.

With embodiments of the instant invention cell motility and changes in cell properties can be detected as it occurs, thus circumventing the need to incorporate detection labels such as radiometric, colorimetric, fluorescent labels or the need to use microscopy for evaluation. A calorimetric resonant reflectance biosensor detects directional cell movement and cell attachment as the cells transverse from an area containing no chemoattractant or protein to an area possessing an entity that induces cell motility. Analysis of cellular movement across a biosensor surface can be expediently monitored in real time, in a label free manner. Several other changes in cell growth patterns or other cell changes can be detected using the methods of this invention, such as change in cell morphology, change in cell adhesion, change in cell migration, change in chemotaxis or other cell movement, change in cell proliferation, change in microtubule structure, change in microfilament structure, granule exocytosis, respiratory burst, cell differentiation (e.g., neuronal elongation), fluctuations in adherence, morphological rearrangement, cytoskeletal rearrangement, cellular differentiation, apoptosis and cell death, change in cell absorption properties, cell signaling (e.g., GPCR/chemokine, RTK, ion channel) and protein secretion. A change in cell properties includes anything that changes a cell's size, shape, height and/or surface uniformity. The methods of the invention can also be used to monitor the reaction and response of cells to environmental or chemical stimuli. Cell movement, changes in cell growth patterns, and other cell responses or changes can be detected in real time using the BIND Biosensor™, BIND Reader™, and BIND Scanner™ (e.g., a calorimetric resonant reflectance biosensor system) and corresponding algorithms for quantification and analysis of data. See, e.g., U.S. Pat. No. 6,951,715, U.S. Patent Appl. Publ. 2004/0151626.

The BIND Biosensor™, BIND Reader™, and BIND Scanner™ (e.g., a calorimetric resonant reflectance biosensor system) and corresponding algorithms can be used to obtain high resolution cell images without the use labels and without killing the cells. High resolution images on the scale of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 75, 100, 150, 175, 200, 300 μm or less can be obtained. Previous methods have employed wide-area or low resolution methods that provide essentially a bi-modal readout that provides little detailed information about the cells' mechanism of response. In contrast, the methods of the invention can provide micrometer resolution, and highly detailed information about a single cell, clusters of cells, or confluent populations of cells and any response the cells may have to stimuli. Wide area or low resolution methods of observing cells can require 10,000 to 60,000 cells in a standard 384 cell culture plate well. The high resolution methods of the invention, however, can provide information on less than 10,000 cells in a standard 384 cell culture plate well. For example, less than about 10, 100, 500, 1,000, 2,500, 5,000, 7,500, or 10,000 cells can be monitored using the methods of the invention. In one embodiment, a single cell can be monitored.

The high resolution, label-free signal provides great detail about cells on the surface of the biosensor. A signal may fall because the an area is experiencing a general reduction, may fall because some of the area is experiencing no reduction but a majority of the area is experiencing reduction, or the majority of the area is experiencing a major reduction while a small area is actually increasing in signal. The present invention allows the area being studied to fall within a cell such that focal adhesion points, cell morphology and the like are being determined on the single cell level so that the specific reason that a signal is falling or rising can be determined. Methods of the invention can even determine the strength of the cells' attachment to the biosensor. Therefore, the methods of the invention can provide information on the responses/changes within the cell or cells and can provide information as to the size of the area in which the cell or cells respond.

Methods of the invention are advantageous because they do not require fixing and/or staining of cells for microscopic or colorimetric/fluorimetric evaluation, they allow for continuous, multiple independent readings of the same population of cells in real time, they are quick, they require minimal reagent usage (both volume and type), and they do not require flowing the cells through a counting device. Additionally, the direction and velocity of cell movement or path can be determined in real time.

Methods of the invention allow for continuous monitoring or multiple independent readings of the same population of cells in real time over many days. Cellular changes can be quantified expediently and objectively over longer periods of time in a normal culturing environment (static with proper media). Methods of the invention can also be used synergistically with fluorescent labels to obtain additional, intracellular data from each cell or cell population.

Cell motility and cell properties can be monitored by taking a PWV for one location over several time periods. Alternatively, scans of a receptacle holding the cells, e.g., a microtiter plate well, can be done over several time periods. A receptacle refers to one container and not a collection of containers, e.g., a multiwell plate.

One or more cells can be applied to a location, such as a microtiter well on a surface of a calorimetric resonant reflectance optical biosensor. In one embodiment of the invention, one or more cells or one or more types of cells can be immobilized to a surface of the biosensor by an antibody (or a binding fragment thereof) that specifically binds an adhesion protein such as integrins, selecting, members of the IgSuperfamily, cadherins, syndecans, and ADAMs. See, e.g., Buckley et al., 1998, Mol. Memb. Biol. 15:167. An adhesion protein is located on a cell surface and is important in binding reactions with other cells and the extracellular matrix. A calorimetric resonant reflectance optical peak wavelength value (PWV) for the location is detected. The one or more cells can be incubated for a period of time (e.g., about 1 second, 30 seconds, 1, 2, 5, 10, 20, 30, 45 minutes, 1, 2, 5, 10 or more hours). Prior to the incubation, or after the incubation, or prior to the incubation and after the incubation one or more test reagents can be applied to the one or more cells. The calorimetric resonant reflectance optical PWV for the location can be detected for a second time. If a change in cell growth pattern or cell property occurs then the reflected wavelength of light is shifted as compared to a situation where no change occurs. The first PWV can be compared to the second PWV. A change in the PWV can indicate a change in cell growth pattern or cell properties in the one or more cells. PWVs over several time periods can be determined and compared. PWVs can also be monitored in real time over the entire time of the assay. For example, PWVs can be taken every second or fractions of seconds over the entire time of the assay. PWVs can also be taken every 5 seconds, 10 seconds, 30 seconds, minute, 5 minutes, 10 minutes or every hour over the course of the assay.

“Specifically binds” or “specific for” means that a first antigen, e.g., a polypeptide, recognizes and binds to an antibody with greater affinity than to other, non-specific molecules. A non-specific molecule is an antigen that shares no common epitope with the first antigen. For example, an antibody raised against an antigen (e.g., a polypeptide) to which it binds more efficiently than to a non-specific antigen can be described as specifically binding to the antigen. In one embodiment an antibody or antigen-binding portion thereof specifically binds to a polypeptide when it binds with a binding affinity about K_(a) of 10⁷ l/mol or more. Specific binding can be tested using, for example, an enzyme-linked immunosorbant assay (ELISA), a radioimmunoassay (RIA), or a western blot assay using methodology well known in the art. Ligands and receptors can also specifically bind one another.

One or more antibodies (or binding fragments thereof) that specifically bind one or more adhesion proteins can be immobilized to the surface of the biosensor. One or more adhesion proteins can be added to the biosensor surface such that they are specifically bind to the immobilized antibodies. One or more types of cells are added to the surface of the biosensor before or after the addition of the adhesion proteins or at the same time the adhesion proteins are added to the biosensor surface. The cells can bind to the adhesion protein via a ligand that is specific for the adhesion protein. The adhesion protein can bind to the antibodies immobilized on the surface of the biosensor. Antibodies can directly bind adhesion proteins or antibodies can bind adhesion proteins through ligands fused or covalently or non-covalently bound to an adhesion protein. For example, the ZZ-binding domain of Protein A can be fused or bound to any adhesion protein. The adhesion protein can then bind IgG through the ZZ-binding domain of Protein A.

For example, VCAM-1 is an adhesion protein that is an endothelial ligand for VLA-4 and for integrin α4β7. VCAM-1 can be fused or bound to a ZZ-binding domain from Protein A. The ZZ-binding domain will specifically bind to IgG immobilized on the surface of the biosensor. Cells that express VLA-4 will bind to the VCAM-1 adhesion protein, which is bound to IgG immobilized to the surface of the biosensor. See FIG. 3. Therefore cells expressing an adhesion protein ligand will be immobilized to the biosensor surface.

Alternatively, one or more antibodies (or binding fragments thereof) that specifically bind one or more adhesion proteins can be immobilized to the surface of the biosensor. One or more types of cells that express the one or more adhesion proteins are added to the surface of the biosensor. The one or more adhesion proteins of the cells can bind to the immobilized one or more antibodies (or binding fragments thereof). Therefore, cells expressing adhesion proteins will be immobilized to the surface of the biosensor.

A test reagent can be, e.g., a metal ion such as Mn2+, Mg2+, or Ca2+, or a nucleic acid molecule, a polypeptide, an antigen, another cell type, an antibody fragment, a small organic molecule, or a small inorganic molecule. A small inorganic molecule or small organic molecule can be less than about 1, 5, 10, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 or 300 Da. Small organic or small inorganic molecules can be about 0.1 to about 500 Da, about 1 to about 300 Da, about 1 to about 200 Da, about 1 to about 100 Da, about 1 to about 50 Da, about 1 to about 25 Da, or any range in between about 0.1 to about 500 Da. A test reagent can also be a small molecule library, which can comprise about 5, 10, 25, 50, 100, 500, 1,000, 5,000, 10,000 or more different small molecules. Alternatively, a small molecule library can comprise only one type of small molecule. Cells can also be subjected to a change to a stimulus such as an environmental change (such as change in temperature, pressure or light).

Cells can respond to stimuli differently based upon what specific proteins or ligands the cell is bound. Therefore, the connections or binding events that the cell is actively involved in can affect how a cell will respond to stimuli such as test reagents or stimuli. The instant invention provides a method to simplify the connections or binding events the cell is experiencing during the assay. The only or one of the only binding events or connections the cell is making is between the cell receptor and adhesion protein that is bound to the one or more antibodies on the biosensor surface (or between a cell expressing an adhesion protein ligand and an adhesion protein that is bound to the one or more antibodies on the biosensor surface). Therefore, a more uniform response of the cell to stimuli or test reagents is expected.

Cell growth pattern or property changes at a biosensor location can be detected via the PWVs of the biosensor surface or monitored more generally using a microscope, digital camera, conventional camera, or other visualization apparatus, magnifying or non-magnifying, that utilizes lens-based optics or electronics-based charge coupled device (CCD) technology.

Preferably, the resolution of the lens of the scanner determining the PWV has an about 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 micrometer pixel size. Previous scanners had a pixel size of greater than about 20 micrometers. Assays of the invention can be completed in less than 1, 2, 3, 4, 5, 6, 7, or 8 hours. That is, cell changes in response to, for example, and added reagent can be determined in a time efficient manner.

All patents, patent applications, and other scientific or technical writings referred to anywhere herein are incorporated by reference in their entirety. The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms, while retaining their ordinary meanings. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims. In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

EXAMPLES

VCAM-1, which was fused to the ZZ-binding domain of Protein A, was added to a biosensor having IgG immobilized to its surface. J6 cells, which express VLA-4, were added to the biosensor surface. FIG. 4 shows normalized PWVs over time for J6 cells that were added to the biosensor. The “J6 cells+1 mM Mn²⁺” and “Buffer” lines shown in FIG. 4 both contained J6 cells and 1 mM Mn²⁺. The “J6 cells and EDTA” line contained J6 cells and EDTA. At the 110 minute mark, EDTA was added to the “J6 cells+1 mM Mn²⁺” cells. The amount of binding of cells to the VCAM-1 immobilized to the surface of the biosensor quickly dropped off because EDTA inhibits the ability of Mn²⁺ to mediate VCAM-1 and VLA-4 binding. The results in FIG. 4 demonstrate that the PWV changes and therefore cell binding changes are indeed due to changes in VLA-4 and VCAM-1 binding. 

1. A method of detecting a change in a cell growth pattern comprising: a) immobilizing one or more cells to a location on a surface of a calorimetric resonant reflectance optical biosensor with an antibody or a binding fragment thereof that specifically binds an adhesion protein; b) detecting a calorimetric resonant reflectance optical peak wavelength value (PWV) for the location; c) incubating the one or more cells for a period of time or applying a test reagent to the one or more cells and incubating the one or more cells for a period of time; d) detecting the calorimetric resonant reflectance optical PWV for the location; and e) comparing the PWV of step (b) to the PWV of step (d); wherein a difference between the colorimetric resonant reflectance optical PWV of step (b) in relation to the colorimetric resonant reflectance optical PWV of step (d) indicates a change in the cell growth pattern in the one or more cells.
 2. The method of claim 1 wherein the change in cell growth pattern is a change in cell morphology, change in cell adhesion, change in cell migration, change in cell proliferation, change in cell death, change in microtubule structure, change in microfilament structure, granule exocytosis, respiratory burst, cell differentiation, or a combination thereof.
 3. The method of claim 1, wherein the PWV is detected using a scanner with a lens having a lower limit pixel size of about 1 micrometers to about 15 micrometers.
 4. The method of claim 1, wherein the location on a surface of a colorimetric resonant reflectance optical biosensor is an internal surface of a vessel selected from the group consisting of a microtiter well, microtiter plate, test tube, Petri dish, microfluidic channel, and microarray.
 5. The method of claim 1, wherein the method is completed in less than one hour.
 6. A method of detecting a change in a cell growth pattern comprising: a) adding one or more cells to a colorimetric resonant reflectance optical biosensor, wherein one or more antibodies or a binding fragments thereof that specifically bind one or more adhesion proteins are immobilized on a location on the biosensor surface; b) optionally adding one or more adhesion proteins that specifically bind the one or more antibodies or binding fragments thereof to the biosensor surface; c) detecting a first calorimetric resonant reflectance optical peak wavelength value (PWV) for the location; d) incubating the one or more cells for a period of time or applying a test reagent to the one or more cells and incubating the one or more cells for a period of time; e) detecting a second PWV for the location; and f) comparing the first PWV and second PWV; wherein a difference between the first PWV in relation to the second PWV indicates a change in the cell growth pattern in the one or more cells.
 7. The method of claim 6, wherein the change in cell growth pattern is a change in cell morphology, change in cell adhesion, change in cell migration, change in cell proliferation, change in cell death, change in microtubule structure, change in microfilament structure, granule exocytosis, respiratory burst, cell differentiation, or a combination thereof.
 8. The method of claim 6, wherein the first and second PWVs are detected using a scanner with a lens having a lower limit pixel size of about 1 micrometers to about 15 micrometers.
 9. The method of claim 6, wherein the location on a surface of a calorimetric resonant reflectance optical biosensor is an internal surface of a vessel selected from the group consisting of a microtiter well, microtiter plate, test tube, Petri dish, microfluidic channel, and microarray.
 10. The method of claim 6, wherein the method is completed in less than one hour.
 11. The method of claim 6, wherein the one or more cells expresses an adhesion protein that specifically binds the one or more antibodies or binding fragments thereof.
 12. The method of claim 6, wherein the one or more cells expresses a receptor that specifically binds the one or more adhesion proteins.
 13. A method of detecting a change in a cell growth pattern comprising: a) adding one or more cells to a calorimetric resonant reflectance optical biosensor, wherein one or more antibodies or a binding fragments thereof that specifically bind one or more adhesion proteins are immobilized on a location on the biosensor surface; b) detecting calorimetric resonant reflectance optical peak wavelength values (PWVs) for the location over the whole time of the assay; b) optionally adding one or more adhesion proteins that specifically bind the one or more antibodies or binding fragments thereof to the biosensor surface; d) incubating the one or more cells for a period of time or applying a test reagent to the one or more cells and incubating the one or more cells for a period of time; wherein a difference between the PWVs over the time of the assay indicates a change in the cell growth pattern in the one or more cells.
 14. The method of claim 13, wherein the change in cell growth pattern is a change in cell morphology, change in cell adhesion, change in cell migration, change in cell proliferation, change in cell death, change in microtubule structure, change in microfilament structure, granule exocytosis, respiratory burst, cell differentiation, or a combination thereof.
 15. The method of claim 13, wherein the first and second PWVs are detected using a scanner with a lens having a lower limit pixel size of about 1 micrometers to about 15 micrometers.
 16. The method of claim 13, wherein the location on a surface of a calorimetric resonant reflectance optical biosensor is an internal surface of a vessel selected from the group consisting of a microtiter well, microtiter plate, test tube, Petri dish, microfluidic channel, and microarray.
 17. The method of claim 13, wherein the method is completed in less than one hour.
 18. The method of claim 13, wherein the one or more cells expresses an adhesion protein that specifically binds the one or more antibodies or binding fragments thereof.
 19. The method of claim 13, wherein the one or more cells expresses a receptor that specifically binds the one or more adhesion proteins. 